Gaston, D. | Search bioRxiv

bioRxiv2026

Evaluation of somatic variant calling methods on high coverage tumour-only amplicon sequencing data in a clinical environment

One of the current workhorses of next-generation sequencing in clinical molecular diagnostics laboratories for profiling somatic mutations in tumours are amplicon-based targeted sequencing panels. Many open-source somatic variant callers are available; however, their use in clinical applications remains under explored. Therefore, we integrated outputs of six variant callers (FreeBayes, MuTect2, Pisces, Platypus, VarDict and VarScan) into a Snakemake pipeline and evaluated tumour-only data from the HD789 commercial reference standard sequenced in triplicate on three different sequencing runs using the Illumina AmpliSeq Focus panel on MiSeq and NextSeq 2000. A 1:4 dilution sample was sequenced for evaluating limits of variant detection. The called variants were analysed along depth, allele frequency, and other sequencing metrics. The variant callers were evaluated by their level of concordance and performance on known somatic variants. FreeBayes consistently called the largest number of somatic variants in each sample but also included more potential artifacts. Overall, FreeBayes, VarScan, MuTect2, and Pisces had the best performance on HD789 data.

bioinformatics↗

bioRxiv2018

mTORC1 activity is dispensable for synthesis of KSHV lytic proteins

Herpesvirus genomes are decoded by host RNA polymerase II, generating viral messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These viral mRNAs have 5{square}-m7GTP caps and poly(A) tails that enable assembly of host translation initiation factors (TIFs) required for viral protein synthesis. Here we show that maximal production of viral proteins during lytic replication requires both the canonical host TIF eukaryotic initiation factor 4F (eIF4F) and the alternative host TIF eIF3d. Despite this, production of infectious virions is largely dependent on eIF3d, with eIF4F playing a minor role. Investigating the effects of eIF3d silencing on the host proteome revealed that it is required for accumulation of proteins involved in cholesterol metabolism, as well as the accumulation of intracellular cholesterol during KSHV lytic replication. In KSHV infected and uninfected cells alike, eIF3d supports efficient translation of cholesterol biosynthesis enzymes, including squalene epoxidase (SQLE), whose function is required to support productive lytic replication during cholesterol scarcity. These findings position eIF3d as a critical TIF required to support translation of essential host mRNAs required for KSHV replication. AUTHOR SUMMARYAnimal cells tightly regulate uptake, synthesis, storage, and export of cholesterol to ensure optimal membrane fluidity and permeability. Many enveloped viruses require cholesterol to support membrane fusion events during cell entry and cell surface budding events during assembly and egress. Herpesviruses like Kaposis sarcoma-associated virus (KSHV) require cholesterol to support entry, but its role in post-entry events, including budding and fusion at internal membranes, remains incompletely understood. Here, through our studies of KSHV protein synthesis, we uncovered a new mechanism of cellular control of cholesterol metabolism involving the host translation initiation factor (TIF) eIF3d. We demonstrated that eIF3d was required to support efficient translation of host mRNAs encoding key proteins required for cholesterol biosynthesis and uptake. Using a lytic reactivation model that bypasses the entry step, we observed that productive KSHV lytic replication is accompanied by eIF3d-dependent increases in intracellular cholesterol. Preventing cholesterol synthesis and uptake, or silencing eIF3d, strongly inhibited production of infectious virions. These studies shed new light on the importance of intracellular cholesterol to support KSHV replication, and provide motivation for future studies of how cholesterol influences discrete post-entry steps of KSHV infection.

microbiology↗